HAM: A new epitope-tag for in vivo protein labeling

The ability to metabolically label proteins with ³⁵S-methionine is critical for the analysis of protein synthesis and turnover. Despite the importance of this approach, however, efficient labeling of proteins in vivo is often limited by a low number of available methionine residues, or by deleterious side-effects associated with protein overexpression. To overcome these limitations, we have created a methionine-rich variant of the widely used HA tag, called HAM, for use with ectopically expressed proteins. Here we describe the development of a series of vectors, and corresponding antisera, for the expression and detection of HAM-tagged proteins in mammalian cells. We show that the HAM tag dramatically improves the sensitivity of ³⁵S-methionine labeling, and permits the analysis of Myc oncoprotein turnover even when HAM-tagged Myc is expressed at levels comparable to that of the endogenous protein. Because of the improved sensitivity provided by the HAM tag, the vectors and antisera described here should be useful for the analysis of protein synthesis and destruction at physiological levels of protein expression.     

Iron-sulfur centers in the photosynthetic reaction center complex fromChlorobium vibrioforme. Differences from and similarities to the iron-sulfur centers in Photosystem I

The photosynthetic reaction center complex from the green sulfur bacteriumChlorobium vibrioforme has been isolated under anaerobic conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals polypeptides with apparent molecular masses of 80, 40, 30, 18, 15, and 9 kDa. The 80- and 18-kDa polypeptides are identified as the reaction center polypeptide and the secondary donor cytochromec ₅₅₁ encoded by thepscA andpscC genes, respectively. N-terminal amino acid sequences identify the 40-kDa polypeptide as the bacteriochlorophylla-protein of the baseplate (the Fenna-Matthews-Olson protein) and the 30-kDa polypeptide as the putative 2[4Fe-4S] protein encoded bypscB. Electron paramagnetic resonance (EPR) analysis shows the presence of an iron-sulfur cluster which is irreversibly photoreduced at 9K. Photoaccumulation at higher temperature shows the presence of an additional photoreduced cluster. The EPR spectra of the two iron-sulfur clusters resemble those of F_A and F_B of Photosystem I, but also show significantly differentg-values, lineshapes, and temperature and power dependencies. We suggest that the two centers are designated Center I (with calculatedg-values of 2.085, 1.898, 1.841), and Center II (with calculatedg-values of 2.083, 1.941, 1.878). The data suggest that Centers I and II are bound to thepscB polypeptide.     

Enhanced production of laccase in Trametes vesicolor by the addition of ethanol

In a medium containing 40 g ethanol l^–1, laccase production by Trametes versicolor was 2.6 unit per ml of the supernatant, which was over 20 times higher than that without ethanol. Laccase activity with ethanol was quite comparable to that with the well-known inducers such as veratryl alcohol, xylidine and guaiacol. With other white-rot fungi, Coriolus hirsutus and Grifola frondosa, ethanol had a similar stimulatory effect on laccase production.     

Infrared spectroscopy of proteins

This review discusses the application of infrared spectroscopy to the study of proteins. The focus is on the mid-infrared spectral region and the study of protein reactions by reaction-induced infrared difference spectroscopy.     

Selective staining of human platelet glycoproteins using nitrocellulose transfer of electrophoresed proteins and peroxidase-conjugated lectins

Platelet proteins (0.5-5 micrograms) were electrophoresed in a one-dimensional or an unreduced-reduced, two-dimensional sodium dodecyl sulfate gel system. The separated proteins were then transferred electrophoretically to nitrocellulose and reacted with peroxidase-conjugated lectins. Visualization of specific glycoproteins which bound the lectins was made by the chromogenic reaction catalyzed by peroxidase utilizing 3,3'-diaminobenzidine as the substrate. Wheat germ agglutinin specifically reacted with and allowed the visualization of glycoprotein Ib. Peanut agglutinin also specifically stained glycoprotein Ib after treatment of the nitrocellulose transferred proteins with neuraminidase. Ricinus communis agglutinin I stained thrombospondin, a 260 kDa protein, and factor VIII. Concanavalin A stained mainly glycoproteins IIb, III, IV, and V. Glycoproteins Ia, Ic, IIa, and other minor glycoproteins could be separated by unreduced-reduced, two-dimensional gel electrophoresis and were stained weakly with wheat germ agglutinin conjugates. These techniques were found to be reproducible as well as easily applied to the analysis and identification of platelet glycoproteins, particularly when dealing with a limited amount of platelets.     

Versuch einer Kausalanalyse der geotropischen Reaktionskette im Chara-Rhizoid

Summary In the initial phase of the geotropical reaction of the Chara rhizoid the growth difference postulated by Sievers (1967c) between the physically upper, slightly subapical flank and the lower one is demonstrated. In horizontal exposure the growth of the extreme cell apex is continued, while the growth of the lower flank is inhibited and that of the upper one is promoted. In the end phase the cell apex shows a damped oscillation until it finally reaches the vertical growth direction. The statoliths follow the oscillating growth of the cell tip from one flank to the opposite one until they are statistically equally redistributed in their normal position.—In vertical exposure under reduced turgor pressure the statoliths fall down into the extreme cell apex, where they inhibit the growth of this part of the cell wall, while the subapical wall grows transversally.—It is concluded that the statoliths inhibit the growth of the cell wall area which they cover.—The physical phase of the reaction chain, the susception, is the gravity-induced downward displacement of the statoliths. The physiological phase starts with the diversion of the acropetal transport of the Golgi vesicles to the upper part of the cell, which is caused by the block of statoliths (perception). The greater rate of vesicle incorporation into the upper flank in comparison to the lower one causes the subapical growth difference which results in the curvature (reaction).—In the case of the Chara rhizoid Golgi- and statolith-apparatus function as a self-regulating cellular system.

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Herrn Prof. Dr. Dr. h. c. Kurt Mothes zum 70. Geburtstag.     

Colorectal Cancers Mimic Structural Organization of Normal Colonic Crypts

Colonic crypts are stereotypical structures with distinct stem cell, proliferating, and differentiating compartments. Colorectal cancers derive from colonic crypt epithelia but, in contrast, form morphologically disarrayed glands. In this study, we investigated to which extent colorectal cancers phenocopy colonic crypt architecture and thus preserve structural organization of the normal intestinal epithelium. A subset of colon cancers showed crypt-like compartments with high WNT activity and nuclear β-Catenin at the leading tumor edge, adjacent proliferation, and enhanced Cytokeratin 20 expression in most differentiated tumor epithelia of the tumor center. This architecture strongly depended on growth conditions, and was fully reproducible in mouse xenografts of cultured and primary colon cancer cells. Full crypt-like organization was associated with low tumor grade and was an independent prognostic marker of better survival in a collection of 221 colorectal cancers. Our findings suggest that full activation of preserved intestinal morphogenetic programs in colon cancer requires in vivo growth environments. Furthermore, crypt-like architecture was linked with less aggressive tumor biology, and may be useful to improve current colon cancer grading schemes.     

The Legal Regulation of Floats and Gliders—In Quest of a New Regime?

This article analyzes the legal status of unmanned instruments (particularly, floats and gliders) for observation purposes in the ocean environment. These new kinds of instruments are being deployed by the thousands into the oceans, not the least as part of the Argo Project of the International Oceanographic Commission. Their uncontrolled drifting has raised legal questions, especially when such instruments enter waters subject to the jurisdiction of foreign states. The authors argue that the current international legal framework is insufficient to address the pertinent issues, and that a new legal regime is needed.